Method for extracting a macrolide from biomatter

ABSTRACT

Provided is a method for obtaining a macrolide, especially tacrolimus, from biomatter.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §1.119(e) ofProvisional Application Ser. No. 60/356,959, filed Feb. 13, 2002, and isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of extracting a macrolide, forexample tacrolimus, from biomatter, especially fermentation broth.

BACKGROUND OF THE INVENTION

Macrolides are multi-membered lactone rings having one or more deoxysugars as substituents. Erythromycin, azithromycin, and clarithromycinare macrolides that have bacteriostatic and/or bactericidal activity.

Tacrolimus (FK 506) is also a macrolide antibiotic that is also animmunosuppressive agent. More potent than cyclosporin, tacrolimus has aselective inhibitory effect on T-lymphocytes.

The macrolides are typically obtained by fermentation, althoughsynthetic routes to some are known. The present extraction method offersseveral advantages over the prior art. For example, the entirefermentation broth can be used as starting material for the presentmethod (“whole broth method”) and the use of hydrophobic extractionsolvent results in an efficient extraction yield, leaving behind mostwater-soluble impurities, with removal of mycelium in one step.Concentration under reduced pressure at temperature above 25° C. andreduced pressure results in a high evaporation rate of solvent withoutprecipitation or decomposition of tacrolimus. Further advantages of thepresent invention will be apparent to the skilled artisan.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for obtaining amacrolide, especially tacrolimus, including the step of extractingmacrolide-containing biomatter with a hydrophobic extraction solvent,especially wherein the hydrophobic extraction solvent is selected fromthe group consisting of n-butanol, iso-butanol, C₂-C₆ linear andbranched esters of acetic acid or formic acid, C₃-C₆ linear or branchedaliphatic ketones, halogenated methanes, halogenated ethanes, andaromatic hydrocarbons that are liquid at 25° C. and that have a boilingpoint at atmospheric pressure less than about 150° C., to obtain asolution of the macrolide in the hydrophobic extraction solvent, whereinthe pH of the biomatter being extracted is about 5.5 to about 13.

In another aspect, the present invention relates to a method forobtaining a macrolide, especially tacrolimus, including the step ofextracting macrolide-containing biomatter with a hydrophobic extractionsolvent, especially wherein the hydrophobic extraction solvent isselected from the group consisting of n-butanol, iso-butanol, C₂-C₆linear and branched esters of acetic acid or formic acid, C₃-C₆ linearor branched aliphatic ketones, halogenated methanes, halogenated ethanesand aromatic hydrocarbons that are liquid at 25° C. and that have aboiling point at atmospheric pressure less than about 150° C., whereinthe extraction is at a temperature between about 2° C. to about 70° C.,especially between about 15° C. and about 35° C., to obtain a solutionof the macrolide in the hydrophobic extraction solvent.

In a further aspect, the present invention relates to a method forobtaining a macrolide, especially tacrolimus, including the step ofextracting macrolide-containing biomatter with a hydrophobic extractionsolvent, especially wherein the hydrophobic extraction solvent isselected from the group consisting of n-butanol, iso-butanol C₂-C₆linear and branched esters of acetic acid or formic acid, C₃-C₆ linearor branched aliphatic ketones, halogenated methanes, halogenatedethanes, and aromatic hydrocarbons that are liquid at 25° C. and thathave a boiling point at atmospheric pressure less than about 150° C.,wherein the extraction is at a temperature between about 2° C. to about70° C., especially between about 15° C. and about 35° C., and at a pH ofbetween about 5.5 and about 13, especially between about 7.5 and about13, to obtain a solution of the macrolide in the hydrophobic extractionsolvent.

In a further aspect, the present invention relates to a method forobtaining a macrolide, especially tacrolimus, including the steps ofextracting macrolide-containing biomatter with a hydrophobic extractionsolvent, especially wherein the hydrophobic extraction solvent isselected from the group consisting of n-butanol, iso-butanol, C₂-C₆linear and branched esters of acetic acid or formic acid, C₃-C₆ linearor branched aliphatic ketones, halogenated methanes, halogenatedethanes, and aromatic hydrocarbons that are liquid at 25° C. and thathave a boiling point at atmospheric pressure less than about 150° C.,wherein the extraction is at a temperature between about 2° C. to about70° C., especially between about 15° C. and about 35° C., and at a pH ofbetween about 5.5 and about 13, especially between about 7.5 and about13, to obtain a solution of the macrolide in the hydrophobic extractionsolvent, concentrating the macrolide-containing solution, treating theconcentrated solution by column chromatigraphy to obtain at least onemacrolide-containing fraction that is a macrolide-containing solution,and crystallizing the macrolide from the solution.

In yet another aspect, the present invention relates to a method forobtaining a macrolide, especially tacrolimus, including the steps ofextracting macrolide-containing biomatter with a hydrophobic extractionsolvent, especially wherein the hydrophobic extraction solvent isselected from the group consisting of n-butanol, iso-butanol, C₂-C₆linear and branched esters of acetic acid or formic acid, C₃-C₆ linearor branched aliphatic ketones, halogenated methanes, halogenatedethanes, and aromatic hydrocarbons that are liquid at 25° C. and thathave a boiling point at atmospheric pressure less than about 150° C., toobtain a solution of the macrolide in the hydrophobic extractionsolvent, separating the solution containing the macrolide from theextracted macrolide-containing biomatter, concentrating the separatedmacrolide-containing solution, treating the concentrated solution bycolumn chromatography to obtain at least one macrolide-containingfraction that is a macrolide-containing solution, optionallyconcentrating the solution, and crystallizing the macrolide from theoptionallyconcentrated separated solution by cooling, especially to atemperature of about 20° C. or less, and isolating the crystallizedmacrolide.

In yet another aspect, the present invention relates to a method forobtaining a macrolide, especially tacrolimus, including the steps ofextracting macrolide-containing biomatter with a hydrophobic extractionsolvent, especially wherein the hydrophobic extraction solvent isselected from the group consisting of n-butanol, iso-butanol, C₂-C₆linear and branched esters of acetic acid or formic acid, C₃-C₆ linearor branched aliphatic ketones, halogenated methanes, halogenatedethanes, and aromatic hydrocarbons that are liquid at 25° C. and thathave a boiling point at atmospheric pressure less than about 150° C., toobtain a solution of the macrolide in the hydrophobic extractionsolvent, separating the solution containing the macrolide from theextracted macrolide-containing biomatter, concentrating the separatedmacrolide-containing solution, treating the concentrated solution bycolumn chromatography to obtain at least one macrolide-containingfraction that is a macrolide-containing solution, optionallyconcentrating the solution, and crystallizing the macrolide from theoptionally concentrated separated solution by combining the concentratedseparated solution with a crystallization solvent selected fromacetonitrile, methanol, ethanol, acetone, diethyl ether, ethyl acetate,the hexanes, the heptanes, and water, and isolating the crystallizedmacrolide.

In still a further aspect, the present invention relates to a method ofobtaining a macrolide, especially tacrolimus, including the step ofextracting macrolide containing biomatter obtained from a microorganismselected form Streptomyces tsukubaensis, Streptomyces hygroscopicus, andStreptomyces lividans, with a hydrophobic extraction solvent, whereinthe hydrophobic extraction solvent is selected from the group consistingof n-butanol, iso-butanol, C₂-C₆ linear and branched esters of aceticacid or formic acid, C₃-C₆ linear or branched aliphatic ketones,halogenated methanes, halogenated ethanes, and aromatic hydrocarbonsthat are liquid at 25° C. and that have a boiling point at atmosphericpressure less than about 150° C., to obtain a solution of the macrolidein the hydrophobic extraction solvent, wherein the pH of the bimatterextracted is about 5.5 to about 13.

In yet another aspect, the present invention relates to a method ofobtaining tacrolimus from tacrolimus-containing biomatter including thesteps of: extracting tacrolimus-containing biomatter that is wholefermentation broth obtained by fermentation of a microorganism selectedfrom the group consisting of Streptomyces tsukubaensis, Streptomyceshygroscopicus, and Streptomyces lividans, with a hydrophobic extractionsolvent selected form the group consisting of n-butyl acetate, isobutylacetate, t-butyl acetate, ethyl acetate, propyl acetate, ethyl formate,butyl methyl ketone, dichloromethane, chloroform, tetrachloromethane,and toluene at a temperature between about 2° C. and about 70° C.,especially between about 15° C. and about 35° C. at a pH between about5.5 and about 13, especially between about 7.5 and about 13 to obtain asolution of tacrolimus in the hydrophobic extraction solvent; separatingthe tacrolimus-containing solution from the extractedtacrolimus-containing biomatter; concentrating the tacrolimus-containingsolution; treating the concentrated solution by column chromatography toobtain at least one macrolide-containing fraction that is amacrolide-containing solution, optionally concentrating the solution,and crystallizing the tacrolimus from the optionally concentratedseparated solution by cooling it or by combining it with acrystallization solvent selected from acetonitrile, methanol, ethanol,acetone, diethyl ether, ethyl acetate, the hexanes, the heptanes, andwater, whereby a precipitate of crystallized tacrolimus is formed; andseparating tacrolimus.

In still yet another embodiment, the present invention relates to amethod of obtaining tacrolimus from tacrolimus-containing biomatterincluding the steps of: extracting tacrolimus-containing biomatter thatis whole fermentation broth obtained by fermentation of a microorganismselected from the group consisting of Streptomyces tsukubaensis,Streptomyces hygroscopicus, and Streptomyces lividans, with a iso-butylacetate, at a temperature between about 2° C. and about 70° C.,especially between about 15° C. and about 35° C., to obtain a solutionof tacrolimus in iso-butyl acetate solvent; separating thetacrolimus-containing iso-butyl acetate solution from the extractedtacrolimus-containing biomatter; concentrating the tacrolimus-containingiso-butyl acetate solution; treating the concentrated solution by columnchromatography to obtain at least one macrolide-containing fraction thatis a macrolide-containing solution, optionally concentrating thesolution, and crystallizing the tacrolimus from the optionallyconcentrated separated solution by cooling it to a temperature of about20° C. or less, or by combining it with a crystallization solventselected from acetonitrile, methanol, ethanol, acetone, diethyl ether,ethyl acetate, the hexanes, the heptanes, and water, whereby aprecipitate of crystallized tacrolimus is formed; and separatingtacrolimus.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a method for obtaininga macrolide, preferably tacrolimus (also known as FK 506), frommacrolide-containing biomatter that includes the step of extracting themacrolide from the macrolide-containing biomatter with a hydrophobicextraction solvent to obtain a solution of the macrolide in thehydrophobic extraction solvent, wherefrom the macrolide can be obtained.In another embodiment, the present invention provides a method forobtaining a macrolide, preferably tacrolimus, from macrolide-containingbiomatter by extracting the biomatter with a hydrophobic extractionsolvent to obtain a solution of the macrolide followed by concentrationof the solution to obtain a concentrate, wherefrom the macrolide isisolated.

Macrolide-containing biomatter is matter obtained from or through use ofa macrolide-producing microorganism, for example bacteria or fungus thatproduces macrolide by fermentation or culturing or the like.Fermentation of microorganism can be carried out by methods well knownto the skilled artisan and described, for example, in Surjit S. Sengha,Fermentation, in 10 Kirk Othmer Encyclopedia of Chemical Technology, 361(Jacquiline I. Kroschwitz, ed. 4^(th) ed. 1993).

Preferred macrolide-containing biomatter is tacrolimus-containingbiomatter, particularly fermentation broth obtainable by fermentationusing a tacrolimus-producing microorganism, for example, Streptomycestsukubaensis, new and mutated strains thereof, Streptomyceshygroscopicus, and Streptomyces lividans, as described in U.S. Pat. Nos.4,894,366, 5,116,756, 5,624,842, 5,496,727, and 5,622,866.

Mycelium and filtrate obtained by filtration of fermentation broth fromfermentation of a macrolide-producing microorganism are also biomatteruseful in the practice of the present invention. The entire fermentationbroth, i.e. “whole broth” from fermentation of a macrolide-producingmicroorganism, unfiltered or purified to separate mycelium, is apreferred macrolide-containing biomatter for the practice of the presentinvention. When whole broth is used, the present method can be referredto as a “whole-broth method”.

The macrolide-containing biomatter is extracted with a hydrophobicextraction solvent that is a solvent for the macrolide, especiallytacrolimus, but that is only sparingly soluble in water at 25° C.Preferred hydrophobic extraction solvents are C₂-C₆ linear and branchedesters of acetic acid or formic acid, for example iso-butyl acetate,C₃-C₆ linear or branched aliphatic ketones, halogenated methanes, forexample dichloromethane, halogenated ethanes, for exampledichloroethane, and aromatic hydrocarbons that are liquid at 25° C. andthat have a boiling point at atmospheric pressure less that about 150°C. Normal and iso-butyl alcohols can also be used as the hydrophobicextraction solvent.

Preferred as hydrophobic extraction solvents are iso-butyl acetate,n-butyl acetate, t-butyl acetate, ethyl acetate, propyl acetate, ethylformate, butyl methyl ketone (2-hexanone), dichloromethane, chloroform,tetrachloromethane, and toluene.

Ethyl acetate and iso-butyl acetate are particularly preferredhydrophobic extraction solvents.

The extraction to form a solution of the macrolide can be performedusing such methods and equipment as are known to skilled artisan androutiner alike. The method and equipment chosen must only provideadequate agitation and allow for separation of the solution fromextracted macrolide-containing biomatter, or for transfer of theextraction mixture to a separation device. Extraction can be carried outat any convenient temperature between about 2° C. and about 70° C.Preferably, the extraction is carried out at a temperature between about15° C. and about 35° C. The skilled artisan will know to optimize theextraction time depending on the macrolide-containing biomatter,hydrophobic extraction solvent, equipment, and temperatures used. At theend of the extraction, the extraction mixture includes a solution of themacrolide in the hydrophobic extraction solvent as well as residualextracted macrolide-containing biomatter.

In a preferred embodiment, the extraction is performed on biomatter, forexample fermentation broth, that is not first subjected to anypurification treatment, for example filtration that would removemycelium. In this case, the extraction is referred to as whole brothextraction.

The extraction can be performed at any pH between about 1 and about 13.Preferably, the extraction is conducted at a pH between about 5.5 andabout 13, most preferably between about 7.5 and about 13. The pH of thebiomatter, especially fermentation broth, can be adjusted using asuitable inorganic base, for example NH₄OH, NaOH, KOH, LiOH, or Ca(OH)₂,to mention just a few. The present inventors have observed particularadvantages, especially in regards to the purity of macrolide, when theextraction is carried-out on biomatter having a pH between about 5.5 andabout 13. Preferably, the pH is an alkaline pH, especially a pH betweenabout 7.5 and about 13.

Following extraction, the solution of macrolide in hydrophobicextraction solvent is separated from the extraction mixture and, inpreferred embodiments, concentrated to obtain a concentrate. Theseparation can be accomplished using methods and equipment well known toskilled artisan and routiner alike, for example decanting, separating ina separatory funnel, and centrifuging using a liquid-liquid centrifuge.

The macrolide-containing solution separated from extractedmacrolide-containing biomatter is concentrated to obtain a concentrate.The concentration can be at prevailing atmospheric pressure (which theskilled artisan recognizes varies slightly about a mean of 760 mm Hg),or it can be at reduced pressure, attained with the aid of, for example,a vacuum pump or water aspirator. The concentration is preferablycarried out at a temperature above about 25° C. The concentration iscarried out until the volume of the macrolide-containing solution isreduced to about 0.2 to about 2.0 percent of its initial volume, or lessto provide concentrated macrolide-containing solutions (“concentrate”).Crude tacrolimus can be isolated from the concentrate.

In preferred embodiments, the concentrate is treated by columnchromatography on a silica gel column. The chromatography method appliedcan be that described in U.S. Pat. No. 4,894,366, incorporated herein inits entirety by reference.

For treatment, concentrate (concentrated macrolide-containing solution)is loaded onto a silica gel column. For loading, the concentrate can becombined with a solvent that is a solvent for the macrolide, for exampleethyl acetate, and slurried with silica gel. Solvent is removed from theslurry to afford silica gel loaded with macrolide and other substancesfrom the concentrate. The loaded silica gel is charged to the top of thecolumn and, if desired, a loading eluent is passed through the column.The column is then eluted with an eluent.

The eluent can be isochratic, that is of constant composition, or thecomposition of the eluent can be varied during elution. Preferredeluents include mixtures of ethyl acetate and hexane.

Fractions are collected to obtain at least one macrolide-containingfraction that is a solution of macrolide in eluent. Multiplemacrolide-containing fractions can be combined to a sinflemacrolide-containing fraction. The macrolide-containing fraction(s) canbe concentrated to obtain a concentrated solution (concentratedfraction) of macrolide, wherefrom the macrolide can be crystallized.

The macrolide is crystallized (precipitated) from the preferablyconcentrated macrolide-containing solution. Macrolide so crystallized(precipitated) can be isolated by, for example, filtration orcentrifugation. Crystallization can be effected by cooling themacrolide-containing solution, preferably to temperature of about 20° C.or less. Crystallization can also be effected with the aid of acrystallization solvent that is combined with the preferablyconcentrated macrolide-containing solution. In preferred embodiments,the combination is thereafter maintained for a holding period of about10 to about 60 hours at a temperature of about 25° C. or below. Thetypical hold time is 48 hours. Useful crystallization solvents includeacetonitrile, methanol, ethanol, acetone, diethyl ether, ethyl acetate,hexanes, heptanes, and water.

In particular embodiments, the macrolide-containing solution isconcentrated to dryness and the macrolide is obtained without furthercooling and without use of a crystallization solvent.

The practice of the invention is further illustrated with the followingnon-limiting examples.

EXAMPLE 1

Fermentation broth (50 ml) was mixed with 50 ml iso-butyl acetate. ThepH of the mixture was adjusted to pH 2 with diluted sulfuric acidsolution. After 30 minutes stirring, phases were separated. Theseparated iso-butyl acetate phase (39 ml) was concentrated to drynessunder reduced pressure at 60° C. The achieved yield was 83%.

EXAMPLE 2

Fermentation broth (50 ml) was mixed with 50 ml iso-butyl acetate.Magnesium sulfate of 250 mg and some drops of diluted dodecyl trimethylammonium chloride solution were added to the mixture. The pH of themixture was adjusted to pH 4 with diluted sulfuric acid solution. After30 minutes stirring, phases were separated. The separated iso-butylacetate phase (44 ml) was concentrated to dryness under reduced pressureat 70° C. The achieved yield was 88%.

EXAMPLE 3

Fermentation broth (50 ml) was mixed with 50 ml iso-butyl acetate.Magnesium sulfate of 250 mg and some drops of diluted dodecyl trimethylammonium chloride solution were added to the mixture. The pH of themixture was adjusted to pH 8 with diluted sodium hydroxide solution. Thecombination was heated to 35-40° C. Phases were separated after 30minutes stirring. The iso-butyl acetate phase (44 ml) was concentratedto dryness under reduced pressure at 82° C. The achieved yield was 94%.

EXAMPLE 4

Fermentation broth (50 ml) was mixed with 50 ml iso-butyl acetate.Magnesium sulfate of 250 mg and some drops of diluted dodecyltrimethyl-ammonium chloride solution were added to the mixture. The pHof the mixture was adjusted to pH 10 with diluted sodium hydroxidesolution. The combination was cooled to 15° C. Phases were separatedafter 30 minutes stirring. The iso-butyl acetate phase (43 ml) wasconcentrated to dryness under reduced pressure at 55° C. The achievedyield was 92%.

EXAMPLE 5

Fermentation broth (50 ml) was mixed with 50 ml ethyl acetate. Magnesiumsulfate of 250 mg and some drops of diluted dodecyl trimethyl ammoniumchloride solution were added to the mixture. The pH of the mixture wasadjusted to pH 4 with diluted sulfuric acid solution. Phases wereseparated after 30 minutes stirring. The separated ethyl acetate phasewas concentrated to dryness under reduced pressure at 29° C. Theachieved yield was 92%.

1. A method for obtaining a macrolide comprising the step of extracting macrolide-containing biomatter that is whole fermentation broth with a hydrophobic extraction solvent to obtain a solution of the macrolide in the hydrophobic extraction solvent, wherein the pH of the macrolide-containing biomatter being extracted is between 8 and about 13 and recovering said macrolide from said solution.
 2. The method of claim 1 wherein the hydrophobic extraction solvent is selected from the group consisting of n-butanol, iso-butanol, C₂-C₆ linear and branched esters of acetic acid or formic acid, C₃-C₆ linear or branched aliphatic ketones, halogenated methanes, halogenated ethanes, and aromatic hydrocarbons that are liquid at 25° C. and that have a boiling point at atmospheric pressure less than about 150° C.
 3. The method of claim 2 wherein the hydrophobic extraction solvent is selected from the group consisting of n-butanol, iso-butanol, n-butyl acetate, iso-butyl acetate, t-butyl acetate, ethyl acetate, propyl acetate, ethyl formate, butyl methyl ketone, methyl iso-butyl ketone, dichloromethane, chloroform, tetrachloromethane, dichloroethane, and toluene.
 4. The method of claim 3 wherein the hydrophobic extraction solvent is ethyl acetate, iso-butyl acetate, or a mixture of these.
 5. The method of claim 4 wherein the hydrophobic extraction solvent is iso-butyl acetate.
 6. The method of claim 1 wherein the extraction is at a temperature between about 2° C. and about 70° C.
 7. The method of claim 6 wherein the extraction is at a temperature between about 15° C. and about 35° C.
 8. The method of claim 1 wherein the pH of the biomatter extracted is between 8 and about
 10. 9. The method of claim 1 wherein the macrolide-containing biomatter is obtained from a microorganism selected from Streptomyces tsukubaensis, Streptomyces hygroscopicus, and Streptomyces lividans.
 10. The method of claim 1 wherein the macrolide is tacrolimus.
 11. The method of claim 10 wherein the pH of the biomatter is between 8 and about
 10. 12. The method of claim 11 wherein the hydrophobic extraction solvent is iso-butyl acetate.
 13. The method of claim 1 further comprising the steps of, after extraction; separating the solution containing the macrolide from the extracted macrolide-containing biomatter to obtain a separated macrolide-containing solution, concentrating the separated macrolide-containing solution to form a concentrated macrolide-containing solution, loading the concentrated macrolide-containing solution onto a silica gel column and eluting with an eluent to obtain at least one macrolide-containing fraction that is a macrolide-containing solution, and isolating the macrolide.
 14. The method of claim 1 wherein the pH of the biomatter is between about 10 and about
 13. 15. The method of claim 1, wherein macrolide is obtained from a macrolide-containing biomatter and the yield of the macrolide is about 83 % to about 94 %.
 16. The method of claim 1 comprising: a) extracting macrolide-containing biomatter that is whole fermentation broth obtained by fermentation of a microorganism selected from the group consisting of Streptomyces tsukubaensis, Streptomyces hygroscopicus, and Streptomyces lividans with a hydrophobic extraction solvent selected form the group consisting of n-butanol, iso-butanol, n-butyl acetate, iso-butyl acetate, t-butyl acetate, ethyl acetate, propyl acetate, ethyl formate, butyl methyl ketone, dichloromethane, chloroform, tetrachloromethane, dichloroethane and toluene to obtain a solution of the macrolide in hydrophobic extraction solvent at a temperature between about 2° C. and about 70° C., wherein the macrolide is tacrolimus.
 17. The method of claim 16, further comprising the steps of: b) isolating the macrolide-containing solution from the extracted macrolide-containing biomatter, c) concentrating the macrolide-containing solution, d) purifying the concentrated macrolide-containing solution on a silica gel chromatography column.
 18. The method of claim 16 wherein the pH of the whole fermentation broth is between 8 and about
 10. 19. The method of claim 16 wherein the hydrophobic extraction solvent is iso-butyl acetate.
 20. The method of claim 16 wherein the extraction is at a temperature between about 15° C. and about 30° C.
 21. A method of obtaining a macrolide from a macrolide-containing biomatter comprising the steps of: a) extracting macrolide-containing biomatter that is whole fermentation broth obtained by fermentation of a microorganism selected from the group consisting of Streptomyces tsukubaensis, Streptomyces hygroscopicus, and Streptomyces lividans with iso-butyl acetate wherein the pH of the macrolide-containing biomatter is between 8 and about 13 to obtain a solution of a macrolide in iso-butyl acetate at a temperature between about 2° C. and about 70° C., b) isolating the macrolide-containing solution from the extracted macrolide-containing biomatter, c) concentrating the macrolide-containing solution, d) purifying the concentrated macrolide-containing solution on a silica gel chromatography column to obtain at least one macrolide-containing fraction that is a macrolide-containing solution, e) concentrating at least one macrolide-containing fraction, wherein the macrolide is tacrolimus, and (f) recovering said tacrolimus from step (e).
 22. The method of claim 21 wherein the pH of the macrolide-containing biomatter is between 8 and about
 10. 23. The method of claim 21 wherein the temperature of extraction is between about 15° C. and about 35° C.
 24. The method of claim 21, wherein macrolide is obtained from a macrolide-containing biomatter and the yield of the macrolide is about 83% to about 94%. 